Controlling Expression of Globin Transgenes at Ectopic Chromosomal Sites: Position effects on the globin genes are a major problem for gene therapy of the hemoglobinopathies and for the creation of animal models that can be used for rigorous comparisons of anti-sickling globins and of anti- beta/S ribozymes. We propose to attempt to solves these problems using Recombinase-Mediated Cassette Exchange (RMCE), a method to perform site-specific chromosomal integration in mammalian cells. In Aim one, we will tackle the problem of transgene silencing in MEL cells and in primary hematopoietic cells. Since the presence of LCR derivatives is clearly not sufficient to prevent positron-effects, we propose to add to our expression cassettes cis-acting elements, such as islands elements, homopolymeric A-T tracts and insulators. Most of these elements are present throughout the genome and are therefore likely to be active at a wide range of ectopic loci. A "stealth" cassette that is entirely free of CpG and are therefore likely to be active at a wide range of ectopic loci. A "stealth" cassette that is entirely free of CpG dinucleotides and that should therefore be invisible to the silencing machinery will also be tested. Because we have demonstrated that positron effects are exquisitely sensitive to local cis-acting elements, we propose to perform all of the above experiments in the context of HIV-derived vector sequences In Aim 2, we will take advantage of RMCE to create an improved sickle cell mouse model using an artificial globin locus containing the alpha, beta and gamma-globin genes. This artificial locus will be optimized for delayed gamma-globin switching and for high-level, balanced expression of the alpha and beta-globin chains. Transgenic animals will then be obtained via insertion of the artificial locus at a predefined and reusable reference ES cells chromosomal site that is favorable for expression in erythroid cells and not subject to position effects. Mice expressing exclusive HbS will be produced through breeding with existing null mutants of the murine alpha and beta-globin loci. This model will be superior to existing ones because it will allow us to test anti-sickling globins by introducing mutations in the coding sequences of the human globin genes, and by inserting the mutated constructs at the same genomic site in the mouse. Because all constructs will be inserted as single copies at a known site, expression levels will be very predictable. Comparison of different anti-sickling or of ribozyme-containing genes will therefore not be impaired by the changes in transgene expression levels that are unavoidable when such experiments are performed using concatemerized randomly integrated transgenes.